MX2010013681A

MX2010013681A - Infection inhibitor.

Info

Publication number: MX2010013681A
Application number: MX2010013681A
Authority: MX
Inventors: Justinus Maria Vlak
Original assignee: Univ Wageningen
Priority date: 2008-06-20
Filing date: 2009-06-19
Publication date: 2011-04-11
Also published as: WO2009153337A1; ECSP11010772A; CN102046650A; BRPI0913246A2

Abstract

The present invention relates to a White Spot Syndrome virus protein and a White Spot Syndrome virus nucleic acid molecule encoding said protein. It also relates to compositions comprising said protein, its use in a vaccine, vaccines comprising said protein and diagnostic tests for the detection of White Spot Syndrome virus specific DNA or antigenic material. Finally it relates to antibodies against said protein.

Description

INFECTION INHIBITOR Description of the invention The present invention relates to the White Spot Syndrome virus protein and a nucleic acid molecule of the White Spot Syndrome virus encoding said protein. It also refers to compositions comprising the protein, its use in a vaccine, vaccines comprising the protein and diagnostic tests for the detection of specific DNA of the White Spot Syndrome virus or antigenic material. Finally, it refers to antibody against this protein.

The white spot syndrome virus (WSSV) is a pathogen of greater economic importance in penaeid shrimp. The virus is not only present a shrimp but also occurs in freshwater and marine crustaceans including crabs and crayfish. In cultured shrimp infection with WSSV can reach cumulative mortality of up to 100% within 3-10 days and can cause large economic losses to the shrimp farming industry. The virus was, for example, found in China in the early nineties, from where it spread rapidly to other areas of shrimp farming in South Asia. WSSV initially appeared to be limited to Asia until it was found in Texas and South Carolina in November 1995. In the beginning from 1999 WSSV was also reported from Central America to South America and has now been detected in Europe. Intensive cultivation of shrimp, inadequate sanitation and international trade has increased the intensity of disease in crustaceans and spread of increased disease. WSSV has become an epizootic disease and is not the only major threat to shrimp farming but also to marine ecology. The disease is O.I.E. mandatory WSSV is a long DNA virus. WSSV virions circulate ubiquitously in the hemolymph of infected shrimp. Electron microscope studies revealed that WSSV virions are particles packed with an ovoid-shaped bacillus shape approximately 275 nm in length and 120 nm in width. The best feature is the appendix in the form of a tail at one end of the virion, in suspension.

The virus particle contains at least 6 major virion proteins of which three (VP664, VP26, VP24 and VP15) are present in the nucleocapsid in the form of a bar and two (VP28 and VP19) reside in the packaging and approximately 40 minor proteins .

The complete sequence of the WSSV genome has been determined (Van Hulten et al., Virology 286; 7.22 (2001), Yang et al., J. Virol. 75; 11811-11820 (2001), Chen et al., Virology 293; 44-53 (2002). DNA has a measure of approximately 300 kbp It contains approximately 180 open reading frames. Most of the proteins that code for genes that do not resemble any of the known proteins or motifs (Van Hulten et al., Virology 286; 7-22 (2001), Yang et al., J. Virol. 74; 11811 -11820 (2001)).

To date, the only possibility to protect shrimp specifically against infection with WSSV is vaccination. The most experimental vaccines are based on the packaging protein of WSSV VP28 (Wittevldt et al., In Fish Shellfish Immunol., 16: 571-579 (2004), Witteveldt et al., In J. Virol. 78: 2057-2061 ( 2004)).

A disadvantage of vaccines in general is that vaccination is a prophylactic action to protect against an infection that may or may not occur. Therefore, if the infection pressure is low, a therapeutic method to solve the problem of infection is more convenient, available on demand and cheaper. For therapeutic purposes, a vaccine is not an appropriate option, because it takes time to build a prophylactic response.

It is an object of the present invention to provide improved or alternative ways to protect shrimp against infection with WSSV, more specifically against oral infection.

Surprisingly, a new gene was found in WSSV, which encodes a novel protein that was shown to play a key role in the primary adjunct of shrimp epithelial cell virus (or more generally, of crustacean). The gene was converted to code the main initial factor in the oral infection process. This will be explained later. A nucleotide sequence of the gene is presented in SEQ ID NO. 1.

It is clear that, if only due to wobble on the second and third bases of many codons, there are some variations in the nucleotide sequence between individual viruses. Accordingly, nucleic acid molecules having at least 90% nucleotide sequence identity with the nucleotide sequence presented in SEQ ID NO. 1 are also considered to represent the new gene according to the invention.

The "% nucleotide sequence identity" of a nucleotide sequence of a nucleic acid molecule with that of a nucleic acid molecule according to the invention can be determined by the alignment of the nucleotide sequence to the total, or a relevant part of the nucleotide sequence of SEQ ID NO: 1. The percent identity between a nucleic acid molecule and a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 can be determined with the computer program "BLAST" 2 SEQUENCES "selecting the sub-program:" BlastN "(T. Tatusova &T. Madden, 1999, FEMS icrobiol.Letters, Vol. 1. 174, p. 247-250), which can be found at the Internet address www.ncbi.nlm.nih.gov/blast/bs2seq/bl2.html. The parameters that must be used are the default parameters: reward for a splice: +1; penalty for a decoupling: -2, penalty for an open interval: 5; extension interval penalty: 2 and interval x_dism¡nution: 50. - '- The BlastN program does not list similarities, only identities: the percentage of nucleotides that are identical as indicated by "identities".

After using computer algorithms to determine the level of identity / decoupling between a nucleic acid and a nucleic acid according to the invention, experimental techniques can also be used. Hybridization is particularly appropriate under conditions of controlled narrowing.

The definition of stringent hybridization conditions, as a function of the identity between two nucleotide sequences, follows from the formula for the melt temperature TM of Meinkith and Wahl (1984, Anal. Biochem., - vol.138, p.267 -284): Tm = [81.5 ° C + 16.6 (log M) + 0.41 (% GC) -0.61 (% formamide) -500 / LJ-1 ° C / 1% -dissociation In this formula: M is the molarity of the monovalent cations; % GC is the percentage of guanosine and nucleotides of cytosine in the DNA; L is the length of the hybrid in base pairs; and "decoupling" is the lack of an identical coupling.

The washing conditions subsequent to hybridization can also be made more or less stringent, thus selecting higher or lower percentages of identity respectively.

In general, the highest narrowness is obtained by reducing the salt concentration, and increasing the incubation temperature. It is within the ability of one skilled in the art to select the hybridization conditions that are coupled to a certain level of percent identity as determined by the computer analysis.

For the purpose of this patent, "stringent conditions" are those conditions under which a nucleic acid molecule hybridizes if it has a decoupling of 10% or less: that is, if it is at least 90% identical to the nucleotide sequence shown in SEC ID NO: 1.

Accordingly, if a nucleic acid molecule hybridizes under stringent conditions to the nucleotide sequence described in SEQ ID NO: 1, it is considered to be a nucleic acid molecule according to the invention.

Therefore, a first embodiment of the present invention relates to a nucleic acid molecule of the White Spot Syndrome virus (WSSV) having a nucleotide sequence according to SEQ ID NO: 1 or a nucleic acid molecule capable of to hybridize under astringent conditions to the nucleic acid molecule having a nucleotide sequence according to SEQ ID NO: 1.

Since the nucleic acid molecule according to the invention is specific for the White Spot Syndrome virus, a diagnostic test based on the nucleic acid molecule is very appropriate for diagnosing the presence or absence of WSSV in water, more specifically in the water of shrimp farm pools. One such test may be a hybridization test as described above. It can be a standard PCR test.

Accordingly, another embodiment of the present invention relates to diagnostic tests for the detection of specific DNA of the White Spot Syndrome virus, characterized in that said test comprises a nucleic acid molecule according to the invention or PCR primers based on the nucleic acid molecule. Standard PCR textbooks provide methods for determining the length of the primers for selective PCR reactions with WSSV DNA according to the invention. Primer fragments with a nucleotide sequence of at least 12 nucleotides are frequently used, but primers of more than 15, more preferably 18 nucleotides are somehow more selective. Especially primers with a length of at least 20, preferably at least 30 nucleotides are very generally applied. The PCR techniques are extensively described in Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN 0-8799-447-5 (1995). Precisely as an example, these primers can, for example, comprise the first 20 and the last 20 nucleotides of the nucleic acid molecule having a nucleotide sequence according to SEQ ID. NO: 1.

The protein encoded by the nucleic acid molecule having a nucleotide sequence according to SEQ ID NO: 1 has an amino acid sequence as demonstrated in SEQ ID NO: 2.

The protein encoded by the gene has a molecular weight of approximately 110 kD. Further analysis of the protein showed that it surprisingly fulfills its role in WSSV which is analogous to the role of the P74 protein in baculoviruses, although the P74 protein is much smaller; approximately 74 kD. The fact that these proteins differ greatly is undoubtedly due to the fact that the baculovirus and WSSV belong to completely different virus families and have a totally different host range.

The novel protein according to the invention would, for reasons listed below, be referred to as WSSP PAP, or briefly PAP (Primary Attachment Protein).

The great difference in MW for WSSV PAP on the one hand, and the similar baculovirus protein P74 on the other hand clearly follows from table 1.

The evolutionary distance of WSSV PAP according to the invention and baculovirus P74 clearly shows from Figure 1. WSSV PAP and baculovirus P74 has no significant sequence.

Table 1: Comparison of the number of amino acids in P74 stick and WSSV PAP, PAP was found to be WSSV analog of Baculovirus P74 protein.

There are some ways in which the protein of the present invention can be used to mitigate or prevent WSSV infection in shrimp.

One scope is to use the protein or a fragment thereof capable of inhibiting the binding between WSSV and crustacean host cells as an inhibitor of infection. The isolated protein is very appropriate as an inhibitor of infection when administered to shrimp, due to the fact that its natural role is to direct the virus to the host cell virus receptor. The main thing is easy: by adding PAP of isolated protein according to the invention, for example, the food of the water, the cellular virus receptors of the shrimp will be occupied by the isolated PAP protein, and in this way blocking the virus receptors of host cell. As a consequence the virus particles can no longer be attached to the host cells; the receiving sites are blocked by PAP.

The PAP protein according to the invention can be successfully used in therapeutic treatments. Especially in regions where the infection pressure is relatively low, farmers may deliberately take the risk and only start treatment as soon as the first clinical signs are manifested. Immediate treatment with full-length isolated protein or a fragment thereof capable of inhibiting WSSV virus attachment to host cells prevents subsequent infection. This has the advantage of saving costs; Costs will be made after the infection actually occurs.

As mentioned above, the protein according to the invention is attached to a viral package. In the live situation, only a part of the protein that protrudes from the surface of the virus plays a role in the attachment. Accordingly, for the purpose of blocking the cell virus receptor, a small fragment of viral protein, since it comprises the part of the protein that is capable of being attached to the cellular virus receptor of shrimp should be sufficient.

Therefore, another embodiment of the present invention relates to a protein or fragment of White Spot Syndrome virus of the protein capable of inhibiting the binding between WSSV and crustacean host cells, wherein the protein or fragment thereof is encoded by an acid molecule nucleic acid having a nucleotide sequence according to SEQ ID NO.:1 or a nucleic acid molecule capable of hybridizing under stringent conditions to the nucleic acid having a nucleotide sequence according to SEQ ID NO. : 1.

In a preferred form of the embodiment, the White Spot Syndrome virus protein according to the invention is a protein having an amino acid sequence according to SEQ ID NO.:1 or a fragment of the shower protein capable of inhibiting the binding between WSSV and crustacean host cells.

As stated above, in principle the isolated complete protein can be used for inhibition. However, a fragment of the protein that is capable of inhibiting the binding between WSSV and the host cells can also be used. This had the advantage that it can often be produced in some easy way, due to the general rule that shorter proteins can be made in expression systems in larger quantities. In addition, the really short peptides of < 50 amino acids can efficiently and very cheaply to make it synthetically.

Another advantage to expressing an appropriate fragment instead of the entire PAP protein is that the fragment can be selected having hydrophilic regions or membrane distribution. This prevents the protein fragment from sticking to the membrane of the cell after expression. Computer programs that predict hydrophilicity are extensively known in the art.

Preferably, a complete protein according is used. However, if you prefer to use a fragment of the protein that is capable of inhibiting the binding between WSSV and host cells, your selection can very easily make a fragment and review a block assay if it inhibits the binding of the virus to epithelial cells.

One such test, in its basic form, comprises the steps of: a) preparing an epithelial tissue explant, for example of stomach or gills, b) incubating the epithelial tissue explant either with full length protein according to the invention (control) or the fragment to be tested c) incubation of the epithelial tissue explant with WSSV, followed by removal of excess WSSV d) incubation of "epithelial tissue explant with conjugated anti-WSSV or anti-VP28 antibodies followed by a staining reaction.

If the protein fragment to be tested is not capable of inhibiting, WSSV will bind to the cells and an enzymatic color reaction with conjugated anti-WSSV or anti-VP28 antiserum will reveal the presence of the virus on the cell surface.

A lack of color reaction indicates a successful inhibitory activity by the fragment to be tested.

Also, as mentioned, it is clear that the PAP protein according to the invention can successfully be used in therapeutic treatments. Especially in regions where the infection pressure is relatively low, farmers can freely take the risk and only start treatment at the first clinical signs. In this way they would save significant costs; Costs will be made after the infection has occurred.

The manner of administering the protein or a fragment thereof according to the invention can be very direct: the protein or fragment thereof can be administered in a pharmaceutical composition comprising the protein or fragment thereof and a pharmaceutically acceptable carrier. Such a carrier can be water or a saline solution such as PBS.

For oral administration the inhibitor is preferably mixed with a suitable carrier for oral administration. In this case, the carrier is preferably a vehicle to which the "protein" adheres, preferably without being covalently bound thereto.These vehicles are, bio-microcapsules, micro-alginates, liposomes and macrosols, cellulose, food or metabolizable substance. such as alpha cellulose or different oils or emulsions of vegetable or animal origin are all known in the art.

An attractive scope is the administration of the protein in high concentrations of living food organisms, followed by the feeding of living food organisms such as brine shrimp to the target animal, for example shrimp. Particularly preferred food carriers Will give oral administration of the vaccine according to the invention are living food organisms that are capable of encapsulating the protein. Another possibility is to express the protein or a fragment thereof according to the invention in. Seaweed yeast, followed by coating of yeast or algae cells in the feed.

Therefore, another embodiment of the present invention relates to a pharmaceutical composition comprising a White Spot Syndrome virus protein or a fragment of the protein according to the invention and a pharmaceutically acceptable carrier.

An alternative scope is also likely, instead of blocking the host cell PAP receptor, it may be preferred to block the viral PAP protein itself. This requires antibodies capable of binding protein PAP of White Monkey Syndrome and at least capable of binding to a fragment of the protein involved in the inhibition of the binding between WSSV and the crustacean host cells. The protein according to the invention or fragments of the same protein capable of inhibiting the binding between WSSV and the crustacean host cells can be used to produce antibodies that can be polyclonal, monospecific or monoclonal (or derivatives thereof). If polyclonal antibodies are desired, techniques for producing and processing polyclonal serum are well known in the art (eg, Mayer and Walter, eds, Immunochemical Methods in Cell and Molecular Biology, Academia Press, London, 1987).

Monoclonal antibodies, reagents against the polypeptide according to the invention (or variants or fragments thereof) according to the invention, can be prepared by immunizing inbred mice by techniques also known in the art (Kohler and Milstein, Nature, 256 , 495-497, 1975).

Methods for large-scale production of antibodies according to the invention are also known in the art. These methods are based on the cloning of (fragments) the genetic information encoding the protein according to the invention into a filamentous phage for phage display. These techniques are described in "Antibody Engineering Page" under "fiL-imentose phage sample" at http: //aximt1.imt.uni-marburq/ / rek / aepphaqe. htl and in the review documents by Cortese, R; et al., (1994) in Trends Biotechn.12: 262-267., by Clackson, T. & Wells, J.A. (1994) in Trends Biotechn.12: 173-183, by Marks, J.D. and col. (1992) in J. Biol. Chem. 267: 16007-16010, by Winter, G. et al. (1994) in Annu. Rev. Immunol. 12: 433-455, and by Little, N. et al (1994) Biotechn. Adv.12. 539-555.

The phages are subsequently used to monitor camelid expression libraries that express heavy chain camelid antibodies. (Muyldermans, S. and Lauwereys, M., Journ.Molec.Acknow.12: 131-140 (1999) and Ghahroudi, M.A. et al., FEBS Letters 414: 512-526 (1997)). The cells of the library expressing the desired antibodies can be replicated and subsequently used for large-scale expression of antibodies.

A very simple and efficient alternative to produce antibody is the production of avian antibodies (egg yolk): IgY. These methods have been described by Schade, R et al., In ATLA 24; 925-934 (1996). The application of the antibodies produced in this way is simple; The egg yolk is, for example, mixed with the food and fed to the shrimp.

Therefore, another embodiment of the present invention relates to antibodies capable of binding to a White Spot Syndrome virus protein or a fragment thereof according to the invention, wherein these antibodies are at least capable of binding to a fragment of the protein involved in the inhibition of the binding between WSSV and crustacean host cells.

With regard to antibodies capable of binding to a PAP protein of White Spot Syndrome virus; As will be discussed below, these antibodies are also appropriate for diagnostic tests. In those tests, there is no requirement for the antibodies to be at least capable of binding to a fragment of the protein capable of being involved in the binding between WSSV and crustacean host cells. For these diagnostic purposes, it is sufficient that the antibodies are capable of binding to the PAP protein of White Spot Syndrome virus. - ^ Accordingly, another embodiment of the present invention relates to antibodies capable of binding to the PAP protein of White Spot Syndrome virus or a fragment thereof.

In regions where the infection pressure is relatively high, it would be desirable to take prophylactic rather than therapeutic measures. This can be done by vaccinating shrimp with the protein or a fragment thereof according to the invention.

Basically, there are two scopes for vaccination; oral vaccination and vaccination through injection. Both vaccination routes have been described for another WSSV.VP28 protein. Therefore, vaccination can be done, for example analogous to vaccination with the WSSV VP28 packaging protein as described by Van Hulten et al., In Virol. 78: 2057-2061 (2005) and Witteveldt et al., In Archives of Virology 150; 1121-1133 (2005).

Both, oral vaccination and vaccination through injection require the production of the protein in accordance with the invention PAP.

For the expression of PAP or fragments thereof, a PAP expression construct fused to (HIS) 6-tag using the vector pET28a (Novagen) can be used. An empty pET28a vector can be used as a control. Both (HIS) 6-PAP and the control vector pET28a can be over expressed according to the manufacturer's instructions. A suitable expression strain is E. coli BL21. When the expression has reached the desired level, the deactivation of the bacteria can be done in formalin for 15 minutes at 20 degrees Celsius.

For the application of oral vaccination, commercial feed granules of approximately 0.02 grams (Coppens International, The Netherlands) can be coated with approximately 108 of the inactivated bacteria. To this extent, the bacterium was washed twice in PBS, re suspended in PBS, mixed with food capsules, incubated on ice for 15 minutes to allow absorption of the bacterial suspension and coated with cod liver oil to prevent dispersal. of the bacterium deactivated in water. Each shrimp can be fed with 8 granules divided into two servings per day. This range is attractive since expression in bacteria is scarce.

Another attractive way to express the PAP protein is, using a baculovirus expression system. This expression system has the advantage because you get high amounts of protein that is more invertebrate in nature.

The coating of the particles can be done in the same way, now using the crude cell preparations. The amount of PAP protein can easily be determined in gel and amounts obtained can be equal to the amounts obtained in a bacterial expression system.

Injection vaccination, analogous to the scope followed for VP28, is preferably done with amounts ranging from 1 to 10 pg of PAP protein in 330 mM NaCl in a final volume of 10 μ. A booster injection with a comparable amount of protein after 5 days increases the level of protection.

Therefore, another embodiment of the present invention relates to a vaccine for combining the infection of White Spot syndrome virus wherein the vaccine comprises a White Spot virus protein or a fragment thereof according to the invention and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier for oral vaccination has been described above. A pharmaceutically acceptable carrier for injection is for example a physiological saline solution or a saline solution.

The WSSV VP28 protein, mentioned above, is known to provide some level of protection, but this level of protection decreases after a while. Accordingly, a vaccine comprising both WSSV PAP and VP "8 of WSSV benefits from the presence of an additional antigen.

Therefore, a preferred form of the embodiment refers to a combination vaccine to combat an infection of White Spot Syndrome virus according to the invention, characterized in that it additionally comprises the VP28 protein of the White Spot Syndrome virus. - As stated above, the present invention describes the novel WSSV PAP protein for the first time. The known WSSV proteins: VP187 and VP110, previously described by Hongyan Li et al., Respectively (Virus Res. 115; 76-84 (2006)), Deng-Feng Li et al., (Virology 368; 122-132 ( 2007)) and Li Li et al. (Journ. Gen. Virol. 87; 1909-1915 (2006)) are also known to play a role, or a different one, in the virus attachment.

It is therefore very attractive to provide a combination vaccine comprising both PAP and VP187, or PAP and VP110, or even PAP, VP110 and VP187.

One such vaccine still interferes at a higher level with the adjunct of WSSV to host cells, since it raises an immune response against two or three viral adjunct proteins. Therefore, another preferred form of this embodiment refers to a combination vaccine to combat the infection of the White Spot Syndrome virus according to the invention, characterized in that it additionally comprises the White Spot Syndrome virus protein VP 110.

Still another preferred form of this embodiment refers to a combination vaccine to combat the infection of the White Spot Syndrome virus according to the invention, characterized in that it additionally comprises a White Spot Syndrome virus protein.

Often, the vaccine is mixed with stabilizers, for example to protect the proteins subject to degradation from being degraded, to improve the expiration of the vaccine, or to improve the efficiency of freeze drying. Useful stabilizers are SPGA (Bovarnik et al., J. Bacteriology 59: 509 (1950)), carbohydrates, for example sorbitol, mannitol, trehalose, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof. , and saline solutions such as alkali metal phosphates. In addition, the vaccine can be suspended in a physiologically acceptable diluent. It is understood that other routes of coadjuvant, add vehicle compounds or diluents, emulsify or stabilize a protein are also included in the present invention.

Vaccines according to the invention which are based on the protein according to the invention or immunogenic fragments thereof can very appropriately be administered in amounts ranging from 1 to 100 micrograms of protein per animal, although smaller doses can be used at the beginning . A dose that exceeds 100 micrograms, would be, although immunologically very appropriate, less attractive for commercial reasons.

Another embodiment refers to the White Spot Syndrome virus protein or a fragment thereof according to the invention for the preparation of a vaccine comprising infection of White Spot Syndrome virus.

The protein according to the invention is also very suitable as a tool in a marker vaccine scope. If it is possible to make WSSV mutants carrying a deletion in the PAP gene and does not comprise the PAP protein according to the invention. This can be done using standard methods such as homologous recombination in crustacean cells after co-transfection with a plasmid loading an elimination of PAP and a marker gene. These mutants have been described for the protein P74 of the crosier virus by Gutiérrez et al., (J. Biotechnol., 116; 135-143 (2205)) with an antecedent given by Faulkner et al., (J. Gen. Virol 78; 3091-'3100 (1997)) and Slack et al., (J. Gen. Virol. 82; 2279-2287 (2001)). Such a mutant can be propagated, for example by injection into river crabs and can be used as a live attenuated vaccine, since it produces the immunogenic protein VP28, but can not cause infection due to lack of PAP.

However, it is important to be able to discriminate between the presence, in a shrimp farm environment, of an attenuated WSSV or WSSV virulent wild type. A test of Such a diagnosis, for example comprising separate wells coated either with antibodies against VP28 or antibodies against PAP can easily do this work. In such a test, the wild-type virus would be attached to the wells comprising VP28 and the wells comprising PAP antibodies, wherein the mutant virus without PAP would be attached only to the wells comprising VP28. A standard color reaction with conjugated anti-WSSV antibodies could be used to monitor the presence / absence of the virus in the respective wells.

Accordingly, another embodiment of the present invention relates to diagnostic tests for the detection of antigenic material of the White Spot Syndrome virus, wherein these tests comprise antibodies against the White Spot Syndrome virus protein according to the invention. with the invention or against a fragment of the PAP protein capable of inhibiting the binding between WSSV and crustacean host cells.

EXAMPLES Example 1 Cloned PAP protein For expression of WSSV PAS or fragments thereof, a PAP expression construct fused to a (HIS) 6-tag using the vector pET28a (Novagen) is used.

The complete PAP protein can be cloned as BamH1 / Pst1 fragment in the vector pET28a after PCR of the WSSV genome using the 5'- and 3 'terminal of 20-mers with the respective additional sites BamHi / Pst1.

Expression of expression in strain E. coli BL21. An empty vector pET28a can be used as a control. Both control vectors (HIS) 6-PAP and pET28a are over expressed in accordance with the manufacturer's instructions.

Example 2 PAP protein expression The His6-PAP and the control proteins are over expressed according to the manufacturer's instructions and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and Western blot with a polyclonal antiserum from WSSV. The bacterial concentration after deactivation is determined with a Beckman DU-7500 photo-spectrometer, where an optical density at 600 nm of 1 equals 109 bacteria per ml. Bacteria are deactivated in 0.5% formalin, incubated for 15 minutes at 20 ° C, checked for deactivation levels, and stored at 4 ° C 'until further use.

Legend to the figure: Figure 1: a tree indicating the evolutionary distance between the proteins involved in virus to host long virus DNA.

AcMNPV = nuclear polyhedrosis virus Autographa califórnica; CpGV = Granulovirus Cydia pomonella; NeleNPV = nuclear polyhedrosis virus Neodiprion lecontii; CuniNPV = Culex nigripalpus nuclear polyhedrosis virus; GbNV = nitrite Gryllus bimaculatus; HzNV = nüdivirus Heliothis zea; GpSGVH = salivary glandular hypertrophy virus Glossina pallipides; MdSGHV = salivary glandular salivary glandular hypertrophy virus; WSSV = White Spot Syndrome Virus.

Claims

1. White spot syndrome virus nucleic acid molecule (WSSV) having a nucleotide sequence according to SEQ ID NO: 1 or a nucleic acid molecule capable of hybridizing under stringent conditions to the molecule of nucleic acid having a nucleotide sequence according to SEQ ID NO: 1.

2. The protein of White Spot Syndrome virus or a fragment of the protein capable of inhibiting the binding between WSSV and crustacean host cells, characterized in that the protein or the fragment thereof is encoded by a nucleic acid molecule according to the claim 1.

3. The protein of the White Spot Syndrome virus according to claim 2, characterized in that it has an amino acid sequence according to SEQ ID NO: 2 or a fragment of the protein capable of inhibiting the binding between WSSV and crustacean host cells.

4'. The pharmaceutical composition comprising a White Spot Syndrome virus protein or a fragment of the protein according to claim 2 or 3, and a pharmaceutically acceptable carrier.

5. The protein of the White Spot Syndrome virus or a fragment thereof according to claim 2 or 3, for use in a vaccine.

6. Use of a White Spot Syndrome virus protein or a fragment thereof according to claim 2 or 3 for the preparation of a vaccine to combat the infection of White Spot Syndrome virus.

7. Vaccine to fight virus infection White Spot Syndrome characterized in that the vaccine comprises a White Spot Syndrome virus protein or a fragment according to claim 2 or 3 and a pharmaceutically acceptable carrier.

8. Combination vaccine to combat the infection of White Spot Syndrome virus according to claim 7, characterized in that it additionally comprises the VP28 protein of the White Spot Syndrome virus.

9. Combination vaccine to combat the infection of White Spot Syndrome virus according to claim 7, characterized in that it additionally comprises the VP110 protein of the White Spot Syndrome virus.

10. Combination vaccine to combat infection of White Spot Syndrome virus according to claim 7, characterized in that it additionally comprises the VP187 protein of the White Spot Syndrome virus.

11. The diagnostic test for the detection of the specific DNA blot syndrome virus characterized in that the test comprises a nucleic acid molecule according to claim 1 or based on the PCR primers based on the nucleic acid molecule.

12. Diagnostic test for the detection of antigenic material of the White Spot Syndrome virus characterized in that the test comprises antibodies against a White Spot Syndrome virus protein or a fragment thereof according to claim 2 or 3.

13. Antibodies capable of "dissociating a protein from White Spot Syndrome virus or a protein shower fragment, characterized in that the protein or fragment thereof is encoded by a nucleic acid molecule according to claim 1.

14. Antibodies capable of binding the White Spot Syndrome virus protein or a fragment according to claim 2 or 3, the antibodies being at least capable of binding to a fragment of the protein involved in the inhibition of the binding between WSSV and cells crustacean hosts.